Fibro-adipogenic progenitors (FAPs) are currently defined by their anatomical position, expression of non-specific membrane-associated proteins, and ability to adopt multiple lineages in vitro. Gene expression analysis at single-cell level reveals that FAPs undergo dynamic transitions through a spectrum of cell states that can be identified by differential expression levels of Tie2 and Vcam1. Different patterns of Vcam1-negative Tie2^high or Tie2^low and Tie2^low/Vcam1-expressing FAPs are detected during neonatal myogenesis, response to acute injury and Duchenne Muscular Dystrophy (DMD). RNA sequencing analysis identified cell state-specific transcriptional profiles that predict functional interactions with satellite and inflammatory cells. In particular, Vcam1-expressing FAPs, which exhibit a pro-fibrotic expression profile, are transiently activated by acute injury in concomitance with the inflammatory response. Aberrant persistence of Vcam1-expressing FAPs is detected in DMD muscles or upon macrophage depletion, and is associated with muscle fibrosis, thereby revealing how disruption of inflammation-regulated FAPs dynamics leads to a pathogenic outcome.

Tissue‐specific transcriptional activators initiate differentiation towards specialized cell types by inducing chromatin modifications permissive for transcription at target loci, through the recruitment of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin‐remodelling complex. However, the molecular mechanism that regulates SWI/SNF nuclear distribution in response to differentiation signals is unknown. We show that the muscle determination factor MyoD and the SWI/SNF subunit BAF60c interact on the regulatory elements of MyoD‐target genes in myoblasts, prior to activation of transcription. BAF60c facilitates MyoD binding to target genes and marks the chromatin for signal‐dependent recruitment of the SWI/SNF core to muscle genes. BAF60c phosphorylation on a conserved threonine by differentiation‐activated p38α kinase is the signal that promotes incorporation of MyoD–BAF60c into a Brg1‐based SWI/SNF complex, which remodels the chromatin and activates transcription of MyoD‐target genes. Our data support an unprecedented two‐step model by which pre‐assembled BAF60c–MyoD complex directs recruitment of SWI/SNF to muscle loci in response to differentiation cues.

Direct generation of a homogeneous population of skeletal myoblasts from human embryonic stem cells (hESCs) and formation of three-dimensional contractile structures for disease modeling in vitro are current challenges in regenerative medicine. Previous studies reported on the generation of myoblasts from ESC-derived embryoid bodies (EB), but not from undifferentiated ESCs, indicating the requirement for mesodermal transition to promote skeletal myogenesis. Here, we show that selective absence of the SWI/SNF component BAF60C (encoded by SMARCD3) confers on hESCs resistance to MyoD-mediated activation of skeletal myogenesis. Forced expression of BAF60C enables MyoD to directly activate skeletal myogenesis in hESCs by instructing MyoD positioning and allowing chromatin remodeling at target genes. BAF60C/MyoD-expressing hESCs are epigenetically committed myogenic progenitors, which bypass the mesodermal requirement and, when cultured as floating clusters, give rise to contractile three-dimensional myospheres composed of skeletal myotubes. These results identify BAF60C as a key epigenetic determinant of hESC commitment to the myogenic lineage and establish the molecular basis for the generation of hESC-derived myospheres exploitable for "disease in a dish" models of muscular physiology and dysfunction.

Cytokine-dependent gene activation critically depends upon the tyrosine phosphorylation (activation) of STAT transcription factors at membrane-bound cytokine receptors. The extent of STAT activation and hence the specificity of signaling is primarily determined by structural complementarity between the SH2 domain of the STATs and the tyrosine-phosphorylated receptor chains. Here, we identified constitutive nucleocytoplasmic shuttling as another mechanism that controls the differential activation of STAT transcription factors. Our analysis of nucleocytoplasmic cycling of STAT1 revealed that the expression of the alternatively spliced transactivation domain and its signal-dependent serine phosphorylation maximized the rate of nuclear export. Export modulation occurred independently of retention factors or the export receptor CRM1, and was observed both before and during stimulation of cells with cytokines. Our data indicated a dual role for the transactivation domain. It enhanced the nuclear retention of activated STAT1, but had the opposite effect on inactivated molecules. Accordingly, and despite their identical receptor recognition, the STAT1 splice variants differed strongly in the amplitude of tyrosine phosphorylation and in the duration of the cytokine signal. Thus, regulated nuclear export determined the cytokine sensitivity of the shuttling STAT1 transcription factors by controlling their availability at the receptor kinase complex.